Graft-versus-host disease (GVHD) is the main cause of stem cell transplant-related mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Regulatory T cells prevent GVHD in preclinical models and early trials. Adoptive transfer of poly-specific Tregs exploits their natural suppressive functions and aims to alter the in vivo balance of T effectors and Tregs. However, this approach may also produce the same broad immunosuppressive effects that are caused by drugs. Experimental rodent models from our lab and others demonstrate that alloantigen-specific Tregs are more effective at preventing GVHD and improving survival than polyspecific Tregs. Our Long-Term Goal is to exploit Tregs to prevent GVHD in humans without suppressing desirable immune responses against infectious pathogens or malignant cells. Given their low frequency in human blood, several groups have explored ex-vivo Treg expansion for therapeutic application and these expanded Tregs retain suppressive activity. In contrast to polyspecific Tregs expanded non-selectively, antigen-specific Tregs produce selective suppression of allo-responses with no effect on third-party responses and facilitate alloantigen-specific tolerance after HSCT and organ grafting in. Before these results find clinical application, early clinical studies are required to address scientific and mechanistic questions and move the field forward. Our central hypothesis is that donor Tregs specific for host alloantigens presented by dendritic cells will prevent GVHD more effectively than current standard immune suppressive drugs, while preserving immunity to viral pathogens and cancer-associated antigens. The objective of this application is to conduct a first-in-human Phase I adoptive immunotherapy trial of allo-specific Tregs for GVHD prevention after HLA- identical sibling HSCT. Important to the potential application of Tregs to human HSCT, is the development of an immune suppressive platform containing rapamycin that selectively permits survival, expansion and suppressive function of Tregs while inhibiting other effector T cells. By trace-labeling the Tregs, we will also assess Treg repopulation and survival after adoptive transfer to allograft recipients.